Project Summary Almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties, and these so called glycans have been implicated as essential mediators of processes such as protein folding, cell signaling, fertilization, embryogenesis, neuronal development, and the proliferation of cells and their organization into specific tissues. Also, overwhelming data supports the relevance of glycosylation in pathogen recognition, inflammation, innate immune responses, and the development of autoimmune diseases and cancer. Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards which are needed for the fabrication of the next generation of microarrays, for the development of analytical protocols to determine exact structures of isolated glycans, for the elucidation of pathways of glycoconjugate biosynthesis, and as immunogens to produce MABs for glycoprotein isolation and visualization. In this application, we propose to develop novel synthetic strategies that can readily provide large libraries of symmetrical and asymmetrical N-glycans. The new methodologies will make use of readily available starting materials and will be sufficiently standardized that many laboratories, including synthesis service units, can adopt these methods. Furthermore, the synthetic principles of the new approaches can easily be applied to the preparation of other classes of glycans such as O-linked glycans and human milk oligosaccharides (HMOs). The new method will employ a symmetrical biantennary glycan that can easily be isolated from egg yolk. Innovative enzymatic transformations will be developed to desymmetrize this glycan. Furthermore, recombinant N-acetylglucosaminyltransferases (MGAT's) will be used to convert a bi-antennary glycan into tri- and tetra-antennary structures. In the latter transformations, chemically modified UDP-GlcNAc donors will be used to temporarily prevent an arm from enzymatic modification. The use of recently developed technology to express recombinant mammalian glycosyltransferases will be a key feature of the new methodology. To validate the robustness of the methodology, it will be applied to the preparation of a library of glycans derived from human upper airway epithelial cells. The resulting glycans will be valuable for the development of the next generation of glycan microarray to probe carbohydrate?protein interactions in the context of this cell type. The scope of the chemoenzymatic methodology will be further extended by the development of methods that can easily provide highly complex asymmetrical glycans that are modified by sulfate esters. An automation platform will be developed to further increase the speed of chemoenzymatic synthesis using novel capture and release strategies. Attention will focus on ion exchange and nickel-mediated histidine binding events for capture of tagged oligosaccharides. A multi-channel liquid handling robot from Chemspeed equipped with a volumetric dispensing system and lyophilizer will be employed as an automation tool. The latter methodology will, at first, be employed for the preparation of O-linked glycans and human milk oligosaccharides.